PROJECT SUMMARY/ABSTRACT O-Linked N-acetylglucosamine (O-GlcNAc) is a monosaccharide that modifies nucleocytoplasmic proteins and plays an essential role in sensing and signaling in cellular processes. Yet, a mechanistic understanding of how O-GlcNAc functions on many of these proteins remains elusive, partly due to difficulties in inducing protein- specific O-GlcNAcylation in cells. The two enzymes that manipulate O-GlcNAc, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), add and remove O-GlcNAc from thousands of proteins by mechanisms that are not yet understood. Thus, manipulation of protein glycosylation is achieved by tailored approaches to individual proteins in a very limited number of expert laboratories, and functional insights to the glycoprotein, where achieved, are typically difficult to integrate with the broader biological system. Recently, we developed a mechanism to manipulate O-GlcNAc stoichiometry on specific proteins in live cells using nanobodies as proximity-directing agents. Nanobodies are small (12?15 kDa), single domain proteins that possess a hypervariable domain with nanomolar binding affinities, similar to that found in antibodies. Fusion of the nanobody to OGT or OGA enabled facile direction of the enzyme to a library of diverse protein targets for induction or reduction of O-GlcNAc occupancy on the protein in cells. The nanobody fusion proteins revealed new functions attributable to the O-GlcNAcylated protein or OGT itself. Implementation of the proximity- directed OGT or OGA fusion proteins only requires transfection of cells by standard molecular biology protocols and detection of O-GlcNAc stoichiometry on the target protein using readily available Western blot or mass spectrometry techniques. With barriers to manipulation of specific O-GlcNAcylated proteins significantly reduced, we aim to build standardized reagents, protocols, and methods for non-expert labs to manipulate glycan stoichiometry on a desired target protein in cells. To accomplish this, we will first optimize the nanobody-OGT system for protein-specific O-GlcNAcylation in vitro and in vivo and develop protocols based on these optimized procedures. These proximity-directed OGT reagents will be complemented by expanded evaluation of the nanobody recognition technology for identification of optimal binding affinity for selective glycosylation of short peptide tags or endogenous protein targets. The evaluated nanobodies will be inserted to standard vectors for ready testing of nanobodies or additional glycan editing enzymes by non-expert labs. Alongside, we will develop nanobody fusions to additional glycan editing enzymes that manipulate additional glycan structures. Finally, we will provide the reagents and protocols to several collaborating labs for trial use, in addition to standard samples, for application to their biological systems of interest. The data and methods produced will be integrated and published with the goal of the method being readily performed in any lab, irrespective of prior expertise in glycoproteomics.